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Assay Tutorials : Jun 1, 2009 (Vol. 29, No. 11)

High-Content Cytotoxicity Analysis

Methodology Detects and Quantitates Multiparametric Data from Individual Cells
  • Paul Wylie
  • ,
  • Mike Janes

Toxicology profiling is a key part of drug discovery and safety as it provides a crucial means of ranking compounds for consideration in drug discovery. Traditionally toxicity testing has been carried out at late-stage drug discovery in animal models.

It is now recognized that early identification of on- and off-target drug effects results in more timely elimination of drugs, which serves to reduce drug development costs. Furthermore, high-throughput in vitro cell-based assays, which have the advantage of speed, reduced reagent usage, and reduced information content, are becoming a more accepted format.

Cell-based assessment of toxicity is best served by a multiparametric approach in order to establish both phenotypic and mechanistic information. There are a number of key cytotoxic phenomena that can be measured including plasma membrane integrity, DNA damage, DNA synthesis, mitotic arrest, and cytoskeletal disruption. 

This tutorial describes the use of TTP LabTech’s Acumen® eX3 microplate laser scanning cytometer to investigate cytotoxicity using a number of high-throughput, cell-based, ready-to-use assay kits and reagents from Invitrogen, part of Life Technologies.

Laser-scanning cytometry, unlike automated fluorescence microscopy, has the ability to rapidly detect and simultaneously quantitate multiparametric data from individual cells. The Acumen eX3 laser scanning cytometer uses multiple scanning lasers (405 nm, 488 nm, 633 nm) for fluorescence excitation, in conjunction with highly sensitive photomultiplier tubes, for the detection of fluorescently labelled cells, hence offers true multiplexing with a theoretical maximum of 12 data-collection channels.

The laser-scanning approach enables whole-well scanning, which results in the generation of improved and more robust data sets. This instrument can perform optimal signal acquisition in plate densities up to 1,536 wells with scan times of <10 minutes/plate.

Validated In Vitro Cell-Based Assays

A number of different cytotoxicity assays were undertaken. Cell viability was assessed in HeLa cells, which were either untreated or treated with 10 µM staurosporine, using the Image-iT® DEAD™ Green viability stain and the HCS NuclearMask™ Deep Red stain.

Both stains are ideal as they are amenable to fixation and permeabilization, thus enabling multiplexing with other biomarkers of toxicity. The Acumen eX3 cytometer permitted whole-well views of untreated and treated cells with both stains (Figure 1), and quantification of this data using the Acumen eX3 software demonstrated that staurosporine treatment induced a clear increase in the number of dead cells with a log EC50 of -5.68 M (2 µM).

The HCS Mitotic Index Kit was used to measure histone H3 phosphorylation (pH3), a sensitive index of mitosis. Cells were either untreated or treated with 500 nM nocodazole. Histograms were constructed from the DNA profiling data and revealed that there were different proportions of cells in G1 (green) and G2/M (red) for the control and treated cells, respectively (Figure 2).

The percentage distribution of cells in G1 or G2 in control and treated cells demonstrated that nocodazole treatment increased the proportion of cells in G2/M phase. Nocodazole treatment resulted in a concentration-dependent change in the percentage of cells in M (pH3), G1 or G2 (DAPI) phases with a log EC50 of -6.92 M (120 nM). Nocodazole-induced phosphorylation of histone H3 with a high Z´ score of 0.88 demonstrating the assay’s screenability.

Cytoskeletal disruption was determined using Alexa Fluor® 488 phalloidin, a high-affinity probe for F-actin. Cells were treated with 10 µM cytochalasin D, a cell permeable inhibitor of actin polymerization that arrests the cells at the G1-S transition. Whole-well scanning demonstrated that treatment with cytochalasin D resulted in significant disorganization of the cytoskeleton (data not shown) with a substantially greater fluorescent intensity of Phalloidin-Alexa Fluor 488; a log EC50 of -6.56 M (275 nM) was determined for cytochalasin D treated cells. The robustness of the assay was demonstrated by the Z´ value of 0.58.

Genotoxicity

Genotoxicity was evaluated by two different assays. The HCS DNA Damage Kit from Invitrogen was used to measure phosphorylation of histone variant, H2AX, which has been identified as an early response of cells to double-stranded DNA breaks. A549 cells were treated with vehicle or 100 µM etoposide for 24 hours before performing the DNA damage assay.

Figure 3A shows a quantitative representation of pH2AX-positive cells detected with secondary antibodies conjugated to either Alexa Fluor 488 dye or Alexa Fluor 647 dye and demonstrates the robustness of the assay with either secondary antibody conjugate (Z´ values of 0.9 and 0.8 for Alexa Fluor 488 and Alexa Fluor 647, respectively). A concentration dependent curve was generated and used to calculate a log EC50  value of -4.89 M (13 µM) for etoposide.

The second genotoxicity assay that was validated on the Acumen eX3 cytometer was Invitrogen’s Click- iT® EdU cell proliferation assay. This assay is superior to the standard bromodeoxyuridine assay as it negates the need for DNA denaturation, thus making the Click-iT EdU assay a more rapid and reliable option. Nascent DNA synthesis and cell proliferation were measured in cells treated with paclitaxel. The data generated demonstrated that paclitaxel could inhibit cell proliferation with a log IC50 of -8.24 M (6 nM) (Figure 3B).

This tutorial demonstrates that with its high-throughput capabilities, the Acumen eX3 laser scanning cytometer is an ideal instrument for performing in vitro, cell-based, high-content drug toxicity profiling. Each of the cytotoxicity assays was performed using protocols that analyzed every cell contained in each well, using instrumentation that was able to capture, normalize, and export data quickly.

All of the Invitrogen assays validated on the Acumen eX3 cytometer were shown to be robust with excellent screenability. Finally each of the reagents was suitable for both fixed and permeabilized cells, which enables efficient multiplex interrogation of off- and on-target effects of compounds for toxicological profiling.